CN103074420B

CN103074420B - L-type PML/RAR alpha fusion gene detection kit and corresponding detection method

Info

Publication number: CN103074420B
Application number: CN201210449468.XA
Authority: CN
Inventors: 李然; 王宏伟; 覃艳红; 陈秀花; 任方刚; 张耀方; 徐智芳; 李莉; 郭海秀; 王晓娟
Original assignee: Shanxi Medical University
Current assignee: Shanxi Medical University
Priority date: 2012-11-12
Filing date: 2012-11-12
Publication date: 2014-08-27
Anticipated expiration: 2032-11-12
Also published as: CN103074420A

Abstract

The invention relates to the blood molecular biology field, and concretely relates to an L-type PML/RAR alpha fusion gene detection kit and a corresponding detection method. The L-type PML/RAR alpha fusion gene detection kit is composed of the following reagents of a functional oxidized grapheme, FITC-labeled single chain DNA with a sequence of 5'-FITC-TGACCTGCCATTGAG-3', a reagent I, formaldehyde, Escin and a PBS buffer, wherein the reagent I is composed of Tris alkali, NaCl, KCl, MgC12, HCl and H2O, an application method comprises the steps of cells culture, incubation liquid preparation by co-incubation with cells, and cells pretreatment and co-incubation of an incubation liquid and cells. The reagent provided by the kit can rapidly and accurately detect that whether certain cell exists the L-type PML/RAR alpha fusion gene or not, the kit has the advantages of sensibility, specificity, simple operation and rapidity, and the kit has important meaning for APL diagnosis and minimal residue monitoring, and the kit can realize recurrence prediction at early stage and clinical treatment guidance.

Description

A kind of L-type PML/RAR alpha fusion gene detection kit and corresponding detection method

Technical field

The invention belongs to blood biology field, be specifically related to a kind of L-type PML/RAR alpha fusion gene detection kit and corresponding detection method.

Background technology

Leukemia is the abnormal clone's property malignant disease of a class hemopoietic stem cell.Leukemia cell in its clone loses the ability of further differentiation and maturation and is stuck in cytocerastic different steps.In marrow and other hemopoietic tissues, a large amount of hyperplasia of leukemia cell are gathered and infiltrate other Organ and tissues, make normal hematopoiesis suppressed simultaneously, and clinical manifestation is that anaemia, hemorrhage, infection and each organ infiltrate symptom.Chromosome abnormalty is one of common cause of leukemia generation.According to leukemia cell's maturity and natural history, leukemia can be divided into acute and chronic two large classes.The cytodifferentiation of acute leukemia is stuck in the early stage, mostly is initiating cell and prorubricyte, and PD is rapid.Wherein acute promyelocytic leukemia (Acute Promyelocytic Leukemia, APL) is a kind of acute leukemia of the severe haemorrhage that often occurs together, and specific chromosome translocation t (15 occurs more than 95% APL; 17) (q22; Q12～21), cause promyelocytic leukemia ((the Promyelocytic leukemia on karyomit(e) No. 15, PML) Retinoic Acid Receptor Alpha on gene and No. 17 karyomit(e)s (Retinoic acid receptor α, RAR α) gene fusion produces PML/RAR alpha fusion gene.According to the inner breakpoint difference of PML gene, can be divided into three kinds of L, S and V-types, wherein L-type PML/RAR alpha fusion gene is main Types, accounts for 70 ~ 80%.Molecular biology research shows, PML/RAR alpha fusion gene is the molecular marker of APL high degree of specificity, is one of important mechanisms of APL patient's morbidity, as APL MICM classification diagnosis according to one of.In recent years, along with the application clinically of vitamin A acid, white arsenic, APL is alleviated (complete reminion, CR) rate completely and is obviously improved, and the case of long-term disease-free survival increases greatly.But leukemic Problems Concerning Their Recurrence is perplexing medical worker always, key reason is in CR patient body, to still have MRD (minimal residual disease MRD) to exist clinically.In onset patient body, leukemia total cellular score is 10 ¹²～l0 ¹³, reach after hematology CR through chemotherapy induction, in body still residual 10 ⁶～l0 ⁸leukemia cell.Therefore, accurately monitor APL minimal residual disease, predicting recurrence, guiding clinical treatment is one of main path improving APL patient's long-term survival rate.

The method that PML/RAR alpha fusion gene detects at present mainly contains chromosome analysis, real-time quantitative RT-PCR, fluorescence in situ hybridization technique etc., but the problems such as specificity is not high, complex operation, testing cost height that these methods all exist, limit its widespread use [Wei Na, Chen Jinghua, Wang Kun etc. hairpin structure DNA probe is for detection of the research [J] of the electrochemical sensor of leukemia PML/RAR alpha fusion gene. analytical test journal, 2008,27 (9): 907-910].So be badly in need of setting up a kind of new detecting method of easy, quick, high specific.

Summary of the invention

The invention provides a kind of L-type PML/RAR alpha fusion gene detection kit and corresponding detection method, object is to realize the detection of a certain this fusion gene of cell.

The present invention adopts following technical scheme to realize: a kind of L-type PML/RAR alpha fusion gene detection kit, consist of the following composition: functionalization graphene oxide, sequence are single stranded DNA, reagent I, formaldehyde, Escin, the PBS damping fluid of the FITC mark of 5 ' FITC-TGACCTGCCATTGAG-3 ', in test kit, each constituent concentration and consumption are in table 1, and the amount of listed reagent is 10 sample reactions:

Table 1

Sequence number	Reagent name	Volume ml	Explanation
				1	Functionalization graphene oxide	0.2	Concentration 2 mg/ml
2	5’FITC-TGACCTGCCATTGAG-3’	2	Concentration is 1 μ mol/l; Purity is HPLC rank; 7 bases of 5 ' end are positioned at PML gene, and 8 bases of 3 ' end are positioned at RARa gene
				3	Reagent I	7.8	?
4	Formaldehyde	1.0	Concentration 10%V/V
				5	Escin	1.0	Concentration 0.05%V/V
6	PBS damping fluid	130	Concentration 0.1mol/L

Wherein the component of reagent I and each constituent concentration, consumption be in table 2,

Table 2

In mentioned reagent, PBS damping fluid is exactly the PBS preparation that uses 0.1mol/L, can directly use, and its composition and proportioning are the common practise of this area; Described reagent I can be prepared by above-mentioned steps; Described formaldehyde, Escin are purchased from Beckman Coulter Inc.: wherein to rise be that cell stationary liquid, Escin are the agent of cell rupture of membranes to formaldehyde; Sequence is that the single stranded DNA of the FITC mark of 5 ' FITC-TGACCTGCCATTGAG-3 ' can be buied from Shanghai Sheng Gong biotechnology company limited.Known, Graphene (Graphene) is monoatomic layer two dimension atomic crystal, its thickness be only 0.35 nm(be about hair diameter 200,000/), it is in the world the thinnest New Two Dimensional nano material, electricity, mechanics and the thermal property of Graphene excellence makes it have important application prospect in fields such as matrix material, sensor, the energy, becomes the focus of nanometer area research in recent years.Applying more Graphene derivative at biomedical sector is mainly graphene oxide (Graphene Oxide, GO), the present invention carries out functionalization to GO, by synthetic to itself and PEG, makes the GO after functionalization have the characteristic such as better biocompatibility, hydrophilic solution stability.The functionalization GO obtaining has very strong adsorptive power to the designed ssDNA probe of the present invention, and can cancellation be marked at the fluorescence on single stranded DNA; When with the present invention in pretreated cell after complementary target molecule effect, the single stranded DNA of fluorescent probe mark can come off from functionalization GO, the fluorescence of probe is restored.

A detection method for L-type PML/RAR alpha fusion gene detection kit, comprises the following steps:

(1) cell cultures: cell to be measured is inoculated in containing in the RPMI-1640 nutrient solution of 10% foetal calf serum, at 37 DEG C, 5%CO ₂under condition, cultivate, within 2 ~ 3 days, half amount is changed liquid; (2) the Incubating Solution preparation of hatching altogether with cell: be that the single stranded DNA of FITC mark of 5 ' FITC-TGACCTGCCATTGAG-3 ' and reagent I are mixed and obtained Incubating Solution by functionalization graphene oxide, sequence; (3) cell pretreatment: A, by cell harvesting to be measured in streaming centrifuge tube, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant; B, then in above-mentioned streaming centrifuge tube, add 100 μ l formaldehyde, mix at once, under room temperature, leave standstill after 10～15min, after adding the piping and druming of 4ml PBS damping fluid to mix in streaming centrifuge tube, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant; C, finally in the streaming centrifuge tube of handling, add 100 μ l Escins, mix, under room temperature, leave standstill after 5 min, after adding 4 ml PBS damping fluids to mix gently in streaming centrifuge tube, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant is for subsequent use; (4) Incubating Solution and cell are hatched altogether: the Incubating Solution that step (2) is obtained adds in step (3) streaming centrifuge tube after treatment, fully mix, after lucifuge, standing 1h, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant, then to after adding 4 ml PBS damping fluids to mix in streaming centrifuge tube again, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant, then add 1ml PBS damping fluid in streaming centrifuge tube, after mixing, carry out interpretation of result.

Analytical procedure in above-mentioned steps (4) adopts laser confocal microscope method and flow cytometer method.

The use principle of test kit of the present invention is: after the mixed solution of the single stranded DNA of pretreated cell to be measured and reagent I, functionalization GO, FITC mark is hatched altogether, the ssDNA probe absorption of functionalization GO and FITC mark, and cancellation is marked at the FITC fluorescence on single stranded DNA, if there is L-type PML/RAR alpha fusion gene to exist in cell to be measured, the single stranded DNA of FITC mark is combined with the mRNA of L-type PML/RAR alpha fusion gene, the single stranded DNA of FITC mark is come off from functionalization GO, occur green fluorescence signal.The present invention analyzes by laser confocal microscope and flow cytometer.

Compared with prior art, the present invention utilizes functionalization GO as carrier and fluorescent quenching instrument, carry fluorescently-labeled single stranded DNA as probe, can fast, accurately detect a certain cell and whether have L-type PML/RAR alpha fusion gene, for the detection of acute promyelocytic leukemia (Acute Promyelocytic Leukemia, APL) PML/RAR alpha fusion gene provides a kind of simple to operate, responsive, special, detection kit fast and effectively.This diagnosis for APL and small residual monitoring thereof have great importance, and can accomplish early prediction recurrence, clinical treatment is had to directive significance.

Brief description of the drawings

Fig. 1 is K562 cell laser confocal microscope analytical results figure mono-;

Fig. 2 is K562 cell laser confocal microscope analytical results figure bis-;

Fig. 3 is NB4 cell laser confocal microscope analytical results figure mono-;

Fig. 4 is NB4 cell laser confocal microscope analytical results figure bis-;

Fig. 5 is not for having FITC labeled ssdna to intervene NB4 cell control group PML/RAR alpha fusion gene flow cytometry analysis result figure;

Fig. 6 is that FITC labeled ssdna is intervened NB4 test cell line group PML/RAR alpha fusion gene flow cytometry analysis result figure.

Embodiment

Below in conjunction with embodiment, the invention will be further described, but the present invention is not limited to the content of embodiment.

A kind of L-type PML/RAR alpha fusion gene detection kit, consist of the following composition: functionalization graphene oxide, sequence are single stranded DNA, reagent I, formaldehyde, Escin, the PBS damping fluid of the FITC mark of 5 ' FITC-TGACCTGCCATTGAG-3 ', in test kit, each constituent concentration and consumption are in table 1, and the amount of listed reagent is 10 sample reactions:

Table 1

Sequence number	Reagent name	Volume ml	Explanation
				1	Functionalization graphene oxide	0.2	Concentration 2 mg/ml
2	5 ^,FITC-TGACCTGCCATTGAG-3 ^,	2	Concentration is 1 μ mol/l; Purity is HPLC rank; 5 ^,Hold 7 bases to be positioned at PML gene, 3 ^,Hold 8 bases to be positioned at RARa gene
				3	Reagent I	7.8	?
4	Formaldehyde	1.0	Concentration 10%V/V
				5	Escin	1.0	Concentration 0.05%V/V
6	PBS damping fluid	130	Concentration 0.1mol/l

Table 2

Component and the consumption of 1 × PBS damping fluid are as shown in table 3, table 3

The detection method of L-type PML/RAR alpha fusion gene detection kit, comprises the following steps:

(1) preparation of functionalization GO

1, the preparation of GO:

1. 1 g graphite is put into beaker, add wherein 0.75 g NaNO ₃, the dense H of 75 ml ₂sO ₄, beaker is placed in to ice bath, and the mixture in beaker is stirred.Slowly in 1 h, in beaker, add 5 g KMnO continuously while stirring ₄, then under ice bath, continue to stir 2 h, then put after stirring at room temperature 4 d, again under ice bath, in beaker, slowly add 2.2 g KMnO ₄, and continue to stir 30 min.

2. temperature is risen to 98 DEG C, in 1 h, in beaker, slowly add the rare H of 140 ml 5% ₂sO ₄, this process still need complete under agitator stirs, and then continues to stir 2 h under 98 DEG C of water-baths.

3. cool the temperature to after 60 DEG C, add 100 ml 30%H ₂o ₂, at room temperature stir 2 h.

4. will centrifugal 10 min of gained liquid 4000 rpm under room temperature, centrifugal 1 time altogether.Abandoning supernatant, stays throw out.

5. to middle gained precipitating species adds centrifugal 8 min of the rare HCl of 480 ml 3% 4000 rpm under room temperature, centrifugal 2 times altogether.Abandoning supernatant, stays throw out.

6. to in middle gained throw out, add sterile purified water repetitive scrubbing, until supernatant liquor pH value partial neutral.

7. will middle gained throw out is collected in beaker, adds 120 ml sterile purified waters, with after ultrasonic 1 h of cell crushing instrument ultrasound probe, gets supernatant liquor after recentrifuge, obtains GO.

2, the functionalization of GO:

Get 5 ml GO, add wherein 3M NaOH, ultrasonic 4 h, neutralize until liquid pH value partial neutral with HCl.And then add 10 mg/ml PEG, ultrasonic 10 min of water-bath.Add EDAC and reach 5mmol/L, ultrasonic 30 min.And then add EDAC to reach 20mmol/L, then stir 12 h, finally add mercaptoethanol and finish reaction.

(2) cell cultures

NB4 cell (experimental group cell), K562 cell (cellular control unit) are inoculated in containing in the RPMI-1640 nutrient solution of 10% foetal calf serum, at 37 DEG C, and 5%CO ₂under condition, cultivate, within 2 ~ 3 days, half amount is changed liquid.Before experiment, carry out cell counting, adjust cell density by requirement of experiment.

(3) the liquid preparation of hatching altogether with cell

Prepare 12 1.5ml EP pipe packing liquid, be total to Incubating Solution preparation with cell and refer to table 5.

Table 4: with cell Incubating Solution preparation altogether

Grouping	No. 1 liquid	No. 2 liquid (lucifuge preparation)	Explanation
				NB4 cell	The mixed solution of reagent I, functionalization GO	The mixed solution of reagent I, functionalization GO, fluorescent mark single stranded DNA	1,3 parts of No. 2 each preparations of liquid
K562 cell	The mixed solution of reagent I, functionalization GO	The mixed solution of reagent I, functionalization GO, fluorescent mark single stranded DNA	1,3 parts of No. 2 each preparations of liquid

Note: the consumption of each sample is: reagent I 780 μ l, fluorescent mark single stranded DNA 200 μ l, functionalization GO 20 μ l

(4) cell pretreatment

1, NB4 cell is collected respectively in 6 streaming centrifuge tubes, and same K562 cell is also collected in 6 streaming centrifuge tubes.Cell count approximately 2 × 10 in each streaming pipe ⁶individual, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant.

2, in 12 centrifuge tubes in above-mentioned 1, in every pipe, add 100 μ l formaldehyde, mix at once, under room temperature, leave standstill after 10～15min, in every pipe, add 4ml PBS(1 ×) after damping fluid piping and druming mixes, centrifugal 5 min of 1 000 rpm under room temperature, abandoning supernatant.

3, in 12 centrifuge tubes handling in above-mentioned 2, in every pipe, add 100 μ l Escins, mix.Under room temperature, leave standstill after 5 min, in every pipe, add 4 ml PBS(1 ×) after damping fluid mixes gently, centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant is for subsequent use.

(5) Incubating Solution and cell are hatched altogether

Liquid in 12 EP pipes in table 4 is joined respectively in (four) 3 in 12 centrifuge tubes after treatment according to the explanation in table 4, fully mix, lucifuge, leave standstill after 1h centrifugal 5 min of 1000 rpm under room temperature, abandoning supernatant.Then in each centrifuge tube, add again 4 ml PBS(1 ×) after damping fluid mixes, centrifugal 5 min of 1 000 rpm under room temperature, abandoning supernatant, in each centrifuge tube, add 1ml PBS(1 ×) damping fluid, mix and move in 12 holes in 24 orifice plates to be detected with pipettor afterwards.

Analytical procedure

This interpretation of result comprises laser confocal microscope method and flow cytometer method

(1) laser confocal microscope method

1,24 orifice plates are placed on laser confocal microscope Stage microscope.

2, click the fluorescent microscope button in FV10-ASW software, close mercury lamp shutter.Meanwhile, click light field button, close halogen lamp shutter.

3, click dye selection button.In dyestuff list, double-click FITC fluorescence dye.

4, click Apply button.

5, click XY Repeat button and start scanning.

6, regulate green (FITC) image.

7, click Stop button and stop scanning.

8, select AutoHV, and select sweep velocity.

9, click XY button and obtain a sub-picture.

10, click SeriesDone button, " 2D View-LiveImage (X) " 2D interface just occurs.

11, preserve image: the image icon showing in right click image manager, select to save as preservation image.

(2) flow cytometer method

1, in 24 orifice plates after laser confocal microscope detects the NB4 enchylema in 6 holes each collect 500 μ l in 6 streaming pipes as loading sample.

2, Samples detection uses CXP acquisition software.

3, start, machine preheating.

4, adjust light path with standard fluorescence microballoon Flow-check, make the half-CV value of each fluorescence channel be less than 2.

5,6 loading samples are placed in loading dish, select FITC fluorescence channel, adjust the laggard row data gathering of voltage conditions.

6, collecting cell.

7, experiment arranges negative control group simultaneously, loading respectively, image data under identical acquisition condition.

8, carry out interpretation of result with CXP analysis software.

Interpretation of result

(1) laser confocal microscope method interpretation of result

Fig. 1 and Fig. 2 are K562 cell, and Fig. 3 and Fig. 4 are NB4 cell; Fig. 1 and Fig. 3 are without the ssDNA probe intervention of FITC mark, and Fig. 2 and Fig. 4 have the ssDNA probe intervention of FITC mark.Visible: equal redgreen fluorescence in Fig. 1, Fig. 2, Fig. 3; And in Fig. 4, there is green fluorescence.Explanatory view 4 cells have L-type PML/RAR alpha fusion gene; Its reason is: after the mixed solution of pretreated NB4 cell and reagent I, functionalization GO, FITC labeled ssdna is hatched altogether, and the ssDNA probe absorption of functionalization GO and FITC mark, and cancellation is marked at the FITC fluorescence on single stranded DNA; If there is L-type PML/RAR alpha fusion gene to exist, FITC labeled ssdna is combined with the mRNA of L-type PML/RAR alpha fusion gene, and FITC labeled ssdna is come off from functionalization GO, occurs green fluorescence signal.Because K562 cell exists without PML/RAR alpha fusion gene, therefore no matter have or not the equal redgreen fluorescent signal of the ssDNA probe intervention of FITC mark, see Fig. 1 and 2.Even if NB4 cell has PML/RAR alpha fusion gene mrna expression, but due to the ssDNA probe intervention without FITC mark, therefore also redgreen fluorescent signal is shown in Fig. 3.

(2) flow cytometer method interpretation of result

FITC labeled ssdna is intervened NB4 cell PML/RAR alpha fusion gene flow cytometry analysis result figure.Fig. 5 is NB4 cell control group, and Fig. 6 is NB4 cell experiment group.The cell count of the control group FITC positive is (1.20 ± 0.20) %, and the cell count of the experimental group FITC positive is (56.00 ± 8.19) %, further Epidemiological Analysis by statistics, the difference of experimental group and control group has significance (F=134.384, P=0.000), see Fig. 5,6, table 5, illustrate that L-type PML/RAR alpha fusion gene positive cell number accounts for more than 56%, can be judged to be the positive.

Table 5:FITC labeled ssdna is intervened NB4 cell PML/RAR alpha fusion gene result

<110> Mountain Western Medicine S University

<120> L-type PML/RAR alpha fusion gene detection kit and corresponding detection method

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<213> people (Homo sapiens)

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Claims

1. a L-type PML/RAR alpha fusion gene detection kit, it is characterized in that consisting of the following composition: functionalization graphene oxide, sequence are single stranded DNA, reagent I, formaldehyde, Escin, the PBS damping fluid of the FITC mark of 5 ' FITC-TGACCTGCCATTGAG-3 ', in test kit, each constituent concentration and consumption are in table 1, and the amount of listed reagent is 10 sample reactions:

Table 1

Wherein the component of reagent I and each composition consumption be in table 2,

Table 2

。